Characterisation of a Rhizobium loti nodulation mutant : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University
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Date
1991
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Massey University
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Abstract
The aim of the project was to characterise the Rhizobiurn loti Nod- Tn5 mutant strain, PN233. The Tn5 insertion had been previously localised to a 7.1 kb Eco RI chromosomal fragment. This fragment was sub-cloned and a Bam HI/Sal I endonuclease restriction map for the region was determined. Hind III digests were utilised to identify the approximate location of the Tn5 233 insertion and those of four other Tn5 insertions (4016, 4019, 4047 and 4053) in the 7.1 kb region. The
233 mutation was found to map to a 1.45 kb Sal I fragment and that of an overlapping 2.8 kb Barn HI fragment.
The 7.1 kb Eco RI fragment and a larger 22.7 kb fragment that encompassed this region, had been cloned into pLAFRl. The construct carrying the 22.7 kb fragment (pPN305) was crossed into four R.l. bv. trifolii strains, each mutant in one of the four common nod genes, A,B,C, and D. The construct was able to complement the nodC mutation indicating the presence of a nodC gene somewhere on the 22.7 kb region.
The mutations 4047 and 4053 had been found to map to either side of the 233 Tn5 insertion. Both insertions affected nodule formation and were thus included in further plant complementation tests. These experiments involved crossing both the pPN305 and a construct bearing the smaller 7.1 kb Eco RI fragment (pPN25) into the R. loti and R.l. bv. trifolii Tn5 mutants. What was unusual about the results was that, while the 7.1 kb fragment was able to complement the mutations, the larger 22.7 kb fragment which encompasses that region could complement PN4047 and PN4053 but was unable to complement the PN233 mutant.
The 2.8 kb Barn HI and 1.45 kb Sal I fragments, to which the 233 insertion was mapped, and that of an adjacent 1.2 kb Sal I fragment, were sub-cloned and then Bal 31 digested in both orientations to create a series of overlapping fragments. These fragments were then sequenced. The data revealed that the 233 Tn5 had inserted into the R. loti node gene. It was determined that the 4047 Tn5 was also located in this gene, slightly upstream of 233, while 4053 had inserted into
the 5'-region of nodI which is downstream of nodC. Nod.A was identified upstream of nodC indicating an arrangement of common nod genes different from the conventional nod.ABeIJ found in other rhizobia. The promoter for these nod genes, the nod box, was located upstream of the nodA gene.
A particularly puzzling aspect of the results is that, while PN4047 is complemented by both pPN305 and pPN25, PN233, which has an insertion in the same gene, could only be complemented by the smaller fragment carried by the pPN25 construct. To explain this result, it is proposed that PN233 is producing a mutant NodC protein and that this, in combination with doubled copies of a gene or genes present elsewhere on the 22.7 kb fragment, is responsible for interfering with complementation in this mutant. Alternatively, it may be that the imbalance of doubled copies of downstream, co-transcribed genes in the presence of one copy of a functional node gene causes complementation failure.
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Keywords
Plant molecular genetics, Rhizobium loti, Genetics, Mutation (Biology)